B and T lymphocytes are the primary effector cells of the immune responses. Both cell classes are considered to derive ultimately from hematopoietic stem cells in the mammalian bone marrow, via progenitor or precursor cells representing distinguishable stages in the differentiation of each class. Mature T cells develop principally in the thymus, presumably from a precursor cell which migrates from the bone marrow to the thymus at an early stage of T lymphocyte development. Numbers of factors are active on mature peripheral B and T cells, including IL-1, IL-2, IL-4, IL-5, interferon gamma, BSF-2, neuroleukin, transforming growth factor beta and IL-7 (EP0314415).
“Interleukin-7” or “IL-7” refers to a mammalian endogenous secretory glycoprotein which is capable of inducing proliferation of bone marrow-derived lymphocyte progenitors and precursors, including the specialized precursors known as pre-B cells. Originally derived from the stromal element of a bone marrow cell line, IL-7 is also secreted by thymic cells, intestinal epithelial cells, keratinocytes and generally all lymphoid tissues. EP0314415 describes mammalian interleukin-7 proteins and DNAs encoding said proteins. The mature human IL-7 (r-hIL-7), exclusive of glycosylation, produced in Escherichia coli, which is described in EP0314415, exhibits a 17,387 Daltons molecular weight and displays a high activity in vitro on specific bioassays based on the proliferation of various lymphocytes populations. Alternative designations for this molecule are “pre-B cell growth factor” and “lymphopoietin-1”. Indeed, originally, IL-7 was disclosed as a cytokine whose principal activity was induction of precursor B cell proliferation (Namen A. E. et al.; Journal of Experimental medicine; 1988; 167:988-1002). IL-7 has more recently been disclosed as being involved in the survival and proliferation of thymocytes (T-Cells) during early stage of T-cell development (Schluns K. S. et al.; Nature Immunology; 2000; 1(5):426-432). Fry and collaborators further identified IL-7 as a potent modulator of thymic-independent T-cell regeneration in a multifactorial action (Fry T. J. et al.; Blood; 2001; 97(6):1525-1533).
IL-7 has thus a great potential therapeutic use in the stimulation of the proliferation of T cell precursors, of antibody-secreting B cells, in the stimulation of antigen driven T-Cell peripheral expansion, and in the production of naïve T-Cells as well as other hematopoietic cell types. A particularly interesting therapeutic use of active IL-7 molecules is for immune reconstitution of lymphopenic patients: patients treated for a cancer, patients having received a bone marrow or a Stem Cell transfer, patients presenting an acquired or genetic immune deficiency, elderly patients or any patients having low CD4 count. Other interests reside in the ability of IL-7 to produce new naïve CD4 T-Cells or to expand specific pools in order to produce or increase specific immune responses (Vaccine enhancement).
In view of its therapeutic potential, there is considerable interest in developing technologies for producing biologically active IL-7 polypeptides and corresponding drug substances. There is much interest in producing IL-7 drug substances or pharmaceutical compositions that comply with the requirements of regulatory authorities and are suitable for efficient therapeutic uses.
A drug substance or pharmaceutical composition aimed at stimulating a global or specific immune reconstitution should be efficient on a long term, which implies that it should not trigger a specific immune reaction against its own active principle.
The present invention now shows, unexpectedly, that the long term activity of recombinant human IL-7 is mostly expressed by a specific 1-4; 2-5; 3-6 conformer. The present invention further shows that efficient drug substances should not only contain the above conformer as the major constituent, but should also be essentially devoid of other conformers or IL-7 molecular variants, previously considered as active products.
Up to the present invention, although the existence of the Cys: 1-4; 2-5; 3-6 conformer was hypothesized through computational modeling based on hypothetical GM-CSF or IL-4 conformation analogy, only IL-7 conformer exhibiting disulfide bridges Cys: 1-6; 2-5; 3-4 had been characterized by tryptic digest and mass spectrometry. This form was thought to constitute the major form of IL-7. In sharp contrast therewith, the invention now proposes new polypeptide products and pharmaceutical compositions which are essentially devoid of such conformer. The invention also discloses advantageous production and purification methods to produce such polypeptides and compositions, which exhibit a high biological activity with reduced risk of undesired response, such as anti-IL-7 immune responses.
The present invention thus discloses improved methods for the preparation and uses of said purified conformer of human IL-7, devoid of the above mentioned product related substances or impurities, which exhibits a particularly advantageous long term in vivo activity, able to favor global or specific immune responses.